The experiments in this proposal combine in vivo studies in Arabidopsis thaliana, where Dr. Schnell will study the influence of mutants of the TOC complex of chloroplast biogenesis, with biochemical studies aimed to elucidate the functional role of various subunits of the TOC complex in membrane translocation. There are 5 specific aims proposed in this grant application. The first aim involves the study of null mutants of Toc34,75 and 159 in A. thaliana and consequences of expressing mutated forms of these proteins. This work is part of an ongoing collaboration with Dr. Kessler at ETH, Zurich, an expert in A. thaliana genetics. The second aim is related to the first one, in that it will use similar methods to explore the function of Toc 132 and Toc 120, two genes they have discovered that appear to be members of the same family of proteins. The third aim focuses on the functional dissection of Toc 159. Specifically it is proposed to study the role of this GTPase in the recognition of pre-proteins that are imported from the cytosol to the chloroplast. It is proposed to search for other proteins that facilitate this recognition by using established methods of protein-protein interaction. Other proteins are part of the TOC complex, and aims 4 and 5 will use in vitro assays to study their potential regulatory role in protein import and their contact map.